Rare cell analysis is important in medical applications, such as for diagnosis of many diseases including cancers. These applications typically require isolation of certain cells of interest which represent only a small fraction of the analyzed sample. For example, rare cells such as circulating tumor cells (“CTC”) are of particular interest in the diagnosis of metastatic cancers. In conventional methods, CTC are isolated from whole blood by first removing red blood cells (“RBC”) by lyses. In a 10 mL blood sample, for example, a few hundred CTC may be separated from about 800,000,000 white blood cells (“WBC”). This requires methods with high separation efficiency and cell recovery rates.
For rare cells to be analyzed by conventional scanning microscopy methods or molecular methods such as next generation sequencing, normal cells (e.g., WBC) must be reduced to a ratio of 200 to 1 normal to rare cells (typically cancer cells) and the sample volume must be reduced from 10 mL to a few hundred microliters or less.
Several approaches have been developed to date to capture, isolate, and enrich rare cells. One approach is to deplete the WBC from a whole blood sample (e.g., negative depletion). Another approach is to enrich the CTC in a whole blood sample (e.g., positive enrichment). Both of the above approaches rely on a variety of techniques, such as magnetic particles, filtration, flow cytometry, and microfluidic channels and chambers to conduct the rare cell analysis.
Recently, a novel approach to the automation of filtration was developed by Gumbrecht of Siemens. The approach is directed to a microfluidic slide format as shown in US 20120315664, which may be combined with a differential pressure hold as shown in WO 2012159821. The system and method allow for automation of membrane filtration with high recovery of rare cells, such as a >99% reduction of WBC and with complete red blood cell (RBC) removal. In addition, for a whole blood sample containing 1000 cancer cells, the ratio of WBC to cancer cells may be decreased from 50,000-100,000:1 to <200:1. Rare cells may then be measured using an immunocytochemistry (ICC) and/or in situ hybridization (ISH) method.
Immunocytochemistry (ICC) methods use antibodies as an affinity partner for a corresponding rare cell. In-situ hybridization (ISH) methods, on the other hand, use a nucleic acid probe as the affinity partner. ICC and ISH may be performed individually or may be combined with one another such that rare cells are targeted by both an antibody and a nucleic acid for enhanced detection. For example, an ISH method is often done in a combination assay with a subsequent or prior ICC method with or without a subsequent cytological morphology by chromogenic dye staining (Hematoxylin & Eosin (H&E)) staining. See Farace F., et al., Detection of circulating tumor cells harboring a unique ALK rearrangement in ALK-positive non-small-cell lung cancer, J. Clin. Oncol. 2013 Jun. 20; 31(18):2273-81. In a combined assay, the ICC reaction may be done and the slide read on as microscope. Next, the slide may be washed to remove antibodies and the ISH reaction is completed. The ISH reaction may be read on the microscope and the images overlapped. When performed, the final H&E staining eliminates the ISH reaction and is also read on the microscope, and the images may be overlapped.
Automation of ICC and/or ISH requires specific method steps to lower the background and increase the signals observed. There are known causes of background noise or low signals (Cytometry 2001, Vol 43(2), p. 101-109) that may lead to false positive results. For example, background noise may arise due to non-specific binding of probe to nucleic acid sequences (which are not desired) or may arise due to non-specific binding of probe to non-nucleic acid material. Causes of lower signaling include non-exposure of the target nucleic acid, non-binding of the probe to the target, and reversal of binding such that the probe is washed away. In addition, background binding may increase with the temperature of the ICC and/or ISH reaction, either due to evaporation causing concentration increases in antibodies and probes or by driving non-specific binding events. Washing and blocking methods are commonly employed but are unable to sufficiently eliminate background.
Further, the combination of ICC and ISH has problems associated therewith. For example, the combination doubles background issues, requires additional heating steps, and adds additional considerations. For example, if ISH is run before ICC, the enzymatic digestion, post-fixation, denaturation, temperatures, and hybridization typically associated with ISH can destroy antigenic determinants and/or interfere with subsequent antibody bonding, thereby causing false negative or providing reduced rare cell detection. On the other hand, if ICC is run first, enzymatic digestion, stringent washing, and hybridization in formamide may break the antibody binding, and wash the antibody from the cell, thereby resulting in false negatives or reduced rare cell detection. This requires increased control of washing and temperature steps.